Amoebidae 79 



reproduction, but the "concentrated artificial spring water" just given 

 has proven superior in our experience. 



Hydrogen-ion concentration oj cultures. Amoeba proteus will culture 

 successfully at widely varying hydrogen-ion concentrations. Good cul- 

 tures have been obtained at pH values anywhere between 4.0 and 8.5. 

 A pH value of 6.6 to 6.8 is generally considered to be optimum. The 

 optimum pH value, however, is probably dependent upon the salt con- 

 tent of the culture. Therefore for ordinary purposes it is unnecessary 

 to take any steps to control the hydrogen-ion concentration of the cul- 

 tures. The great majority of cultures set up as described above will 

 come to an equilibrium at a favorable hydrogen-ion concentration. 



The maintenance of a constant hydrogen-ion concentration for special 

 purposes is rather difficult. The addition of enough buffer salts to main- 

 tain a constant pH value brings the total salt concentration to a value 

 too high for growth of the Amoebae. However, Hopkins and Johnson 

 (1928) by a gradual addition of buffer salts were able to adapt the 

 Amoebae to the increased salt concentration so that a constant pH value 

 was maintained, and at the same time Amoebae grew and reproduced. 

 This procedure should be as follows: To a new culture in which Amoebae 

 have begun to grow and reproduce add each day about 0.5 cc. of Clark 

 and Lubb's phosphate buffer of the desired pH value for each 100 cc. 

 of culture fluid until a total of 5 cc. of the buffer has been added for 

 each 100 cc. of culture fluid. 



Hopkins (1926) used a feeding method for maintaining a constant 

 hydrogen-ion concentration. Amoeba cultures when first set up as 

 described above first become more acid in reaction and then gradually 

 return to the alkaline side of neutrality. If, when in the return of a 

 culture to alkalinity a given pH value is reached which is desired to be 

 maintained as a constant value, one adds a certain amount of fresh 

 sterilized hay or wheat infusion daily, the acid tendency of the fresh 

 infusion will oppose the alkaline tendency of the culture. By measuring 

 the hydrogen-ion concentration daily and adding fresh infusion accord- 

 ingly it is possible to maintain the concentration within a range of 0.2 pH 

 units. 



Temperature. As long as the temperature in the culture is below 25 

 C. they remain in good condition. If the temperature is too low 

 growth is retarded. It is necessary to freeze them before they are 

 seriously injured. About 22 ° C. is perhaps optimum for growth. In 

 summer when room temperature goes above 25 C. it is advisable to keep 

 cultures in a cool basement room or, better, in a cold room where the 

 temperature is maintained at the desired level. 



Light. Direct sunlight is injurious. Growth is just as good in ab- 

 solute darkness as in any light intensity. 



